Different methods for construction of novel binding proteins have been described (Nygren P A and Uhlén M (1997) Curr Opin Struct Biol 7:463-469). One strategy has been to combine library generation and screening or selection for desired properties.
Original AFFIBODY molecules, populations of such molecules and scaffolds of such molecules have been described i.a. in WO 95/19374, the teaching of which is incorporated herein by reference.
For some applications proteins, polypeptides or AFFIBODY molecules, populations of such molecules and scaffolds with improved properties, such as alkali stability, low antigenicity, structural stability, amenability to chemical synthesis and hydrophilicity, are desired.
Alkali Stability
Production of protein pharmaceuticals and biotechnology reagents requires several purification steps to enrich for specific product while removing unwanted contaminants. Affinity purification mediated by proteinaceous affinity matrices such as monoclonal antibodies and Staphylococcal protein A (SpA) enables efficient purification in one step. However, to make this cost-efficient it is desirable to be able to properly regenerate the affinity matrices. This usually involves a procedure known as cleaning-in-place (CIP), wherein agents—often alkaline solutions—are used to elute contaminants.
Alkali stability is also required with molecular imaging tracers to be labeled with the most common SPECT nuclide technetium-99m, and to enable some other types of chemical modifications to be performed.
Low Antigenicity
Protein based pharmaceuticals, such as therapeutic monoclonal antibodies and AFFIBODY molecules, have the potential to elicit undesired immune responses in humans. The main factors contributing to immunogenicity are presence of impurities, protein aggregates, foreign epitopes e.g. new idiotopes, different Ig allotypes or non-self sequences. In addition, cross-reacting immunoglobulin (Ig) interactions will most likely increase the probability of generating a specific T-cell mediated memory immune response against the protein pharmaceutical. To minimize the risk of unwanted interaction with the immune system it is desirable to eliminate existing immune epitopes by protein engineering of the pharmaceutical.
AFFIBODY molecules are derived from staphylococcal protein A (SpA), which is a cell wall associated receptor on the surface of the Gram positive bacterium Staphylococcal aureus. More precisely, SpA is composed of five highly homologous domains all binding to immunoglobulins of many mammalian species including human. Each SpA domain interacts with human Igs in two different ways; either by direct binding to Fcγ including IgG1, IgG2 and IgG4 (Langone J J (1982) Adv Immunol 32:157-252), or by binding to members of the VH3 family (Silverman G J et al (1992) Int Rev Immunol 9:57-78). The common scaffold of original AFFIBODY molecules is identical to domain B of SpA with the exception of the G29A mutation, which was included to increase protein stability and to eliminate a hydroxylamine cleavage site, and the A1V mutation, introduced in a spacer region between domains (Nilsson B et al (1987), Prot Eng 1:107-113). The amino acid residues in SpA that are involved in the interaction with Fcγ and VH3 are well known and have been described in the literature (Graille M et al (2000) Proc Natl Acad Sci USA. 97:5399-5404). A molecular library of different AFFIBODY molecules was constructed by randomizing surface residues at one face of the molecule, including residues known to be involved in the interaction with Fcγ, thereby eliminating the affinity for Fcγ.
Structural Stability
One of the key factors to success for peptide and protein pharmaceuticals is the stability of the protein. Proteins showing high structural stability will most likely functionally withstand chemical modifications and proteolysis both during production as well as within the human body. Moreover, stability will influence the active shelf-life of the peptide or protein pharmaceuticals as well as the active life of the peptide or protein pharmaceutical within the human body.
Amenability to Chemical Synthesis
Researchers have traditionally obtained proteins by biological methods but chemical synthesis of peptides and small proteins is a powerful complementary strategy and is commonly used in structural biology, protein engineering and biomedical research. Chemical synthesis of proteins offers a rapid and efficient way of producing homogenous proteins free of biological contaminants such as DNA impurities and host cell proteins. Furthermore, flexibility is increased since chemical synthesis allows incorporation of unnatural amino acids, chemical modifications and introduction of biochemical and biophysical probes. The success of chemical synthesis of peptides and proteins is dependent on the amino acid sequence of the molecule in question. Certain amino acid residues show low coupling efficiency, meaning that several steps during synthesis need to be optimized which is a time-consuming process with no guaranteed success. In addition, amino acids difficult to efficiently introduce during chemical synthesis will have greater negative impact on protein yield the longer the protein sequence.
Increased Hydrophilicity
For most applications it is desirable that peptides and proteins are highly soluble showing a low tendency to aggregate. Such protein characteristics are especially important when it comes to protein pharmaceuticals. There is a strong positive correlation between protein surface hydrophobicity and low solubility and increased tendency to aggregate.